PROJECT ABSTRACT Cell motility is a common aspect of both health and disease. Motile cells are critical for the immune system, which protects us. Conversely, aberrant cell motility is a hallmark of malignant tumors, which can be lethal. How cells regulate their movements is therefore an important issue. In this application, we explore the genetics of cell motility by experimentally manipulating in zebrafish a key component of the cytoskeleton? the actin-bundling protein L-plastin. For the study of this protein, the zebrafish provides an optimal combination of genetic and cellular features: 1) Actin-bundling proteins are highly conserved in metazoans, and are almost identical in zebrafish and humans. 2) Similar to humans, there is only one copy of the L-plastin gene in zebrafish, facilitating genetic targeting of this locus. 3) Gene-specific mutagenesis in zebrafish is now highly efficient, allowing the creation of multiple, stable lines for any gene of interest. 4) For the study of cell motility, the transparent tissues of zebrafish allow live visualization of both normal and modified cell movements. Knockout studies in zebrafish have been essential for defining the role of single-gene mutations in development and disease. In the last 9 months, we have used the recently developed CRISPR/CAS9 gene-editing technology to generate four mutant alleles of zebrafish L-plastin. We predict that these novel alleles will lead to observable phenotypes in zebrafish embryonic development, immune system function, or susceptibility to cancer. Students will define the role of L-plastin in the early immune system, observe the effect of L-plastin on single cells in vivo, and validate L-plastin as a histological marker for zebrafish tumor cells. As an additional tool for testing our hypotheses, we will produce for the research community the first L-plastin specific reporter lines, allowing direct visualization of expressing cells anywhere in the organism. These experiments will contribute to our understanding of cell motility in health and disease, and the genetic tools produced will be of future use to researchers studying actin bundling and the cytoskeleton.